大动物体内促组织工程骨成骨及血管化手段的研究

目的：探讨组织工程骨能否修复大动物大段负重骨骨缺损,筋膜瓣能否及如何促进组织工程骨体内成骨及血管化的过程。方法：中国青山羊9只作为空白组,制备单侧胫骨2cm的骨膜与肌缺损,缺损内不植入任何填充物,术后行放射性核素骨显像（ECT）、X线检查、组织学方法评价骨缺损自行修复情况。27只中国青山羊根据骨缺损植入物的不同分为3组：单纯材料组（单纯珊瑚羟基磷灰石coral hydroxyapatite,CHAP）、组织工程骨组（CHAP 经诱导分化的骨髓基质干细胞bone marrow stroma cell,BMSc）、筋膜瓣组（筋膜包裹CHAP 经诱导分化的BMSc）。组织工程骨组和筋膜瓣组分别取9只山羊的BMSc体外进行诱导分化,之后与CHAP复合。制备皮下带蒂深筋膜瓣。各组按不同的设计方案分别植入到骨缺损内。术后2、4、8周行ECT检查,4、8、12周行X线检查、组织学（V-G染色）检查,12周行生物力学检查。结果：术后各项检查结果表明,空白组山羊胫骨2cm的骨缺损是其自身无法修复的,因此是一个理想的骨缺损模型。单纯材料组未能修复骨缺损,仅表现出一个缓慢的爬行替代过程;组织工程骨组可基本修复骨缺损,在成骨质量和血管化过程方面表现出较理想的结果;筋膜瓣组修复骨缺损的效果更为满意,成骨质量和血管化程度亦高于组织工程骨组。结论：山羊胫骨2cm缺损模型不能自主成骨,符合骨组织工程实验的要求。组织工程骨具有良好的修复山羊大段骨缺损的能力。筋膜瓣促组织工程骨成骨的作用是通过其促进组织工程骨血管化这一方式实现的。     

山羊胫骨大段骨缺损的组织工程骨修复及放射性核素显像监测

目的探讨组织工程骨对山羊大段骨缺损的修复能力及放射性核素骨显像技术在此过程中的监测效果。方法将27只中国青山羊,分为组织工程骨组、人工骨组和对照组(每组9只)并分别造成左侧胫骨2cm骨缺损。对组织工程骨组缺损区植入珊瑚羟基磷灰石(CHAP)和骨髓基质干细胞(BMSc)复合体,人工骨组植入CHAP,对照组则不植入任何填充物。采用放射性核素骨显像于术后2、4、8周监测各组骨修复情况。结果通过放射性核素骨显像测定的感兴趣区(ROI)计数和T/NT比值显示:对照组在各时间点均未见再血管化的表现及明显的成骨活动;人工骨组随着时间的延长其血管再生的数量和成骨的质量呈现出上升的趋势;组织工程骨组上升趋势更为显著。结论组织工程骨较人工骨具有更佳的修复大动物大段骨缺损的能力,放射性核素骨显像在修复过程中有比较准确的预测效果。     

山羊胫骨大段骨缺损的组织工程骨修复及放射性核素显像监测

目的：探讨组织工程骨对山羊大段骨缺损的修复能力及放射性核素骨显像技术在此过程中的监测效果。方法：将27只中国青山羊，分为组织工程骨组，人工骨组和对照组（每组9只）并分别造成左侧胫骨2cm缺损，对组织工程骨组缺损区植入珊瑚羟基磷灰石（CHAP）和骨髓基质干细胞（BMSc)复合体，人工骨组植入CHAP，对照组则不植入任何填充物，采用放射性核素骨显像于术后2，4，8周监测各组骨修复情况。结果：通过放射性核素骨显像测定的感兴趣区（ROI）计数和T／NT比值显示：对照组在各时间点均未见再管化的表现及明显的成骨活动；人工骨组随着时间的延长其血管再生的数量和成骨的质量呈现出上升的趋势。组织工程骨组上升趋势更为显著。结论：组织工程骨较人工骨具有更佳的修复大动脉大段骨缺损的能力，放射性核素骨显像在修复过程中有比较准确的预测效果。     

带蒂深筋膜瓣促大段组织工程骨成骨效果的观察

目的 研究带蒂筋膜瓣包裹大段组织工程骨的长期成骨效果.方法 新西兰大白兔36只,每只兔双侧桡骨制作1.5 cm骨缺损,将组织工程骨植入骨缺损处,左侧用带蒂筋膜瓣将之包裹作为实验组,右侧不用带蒂筋膜瓣包裹作为对照组.术后早期2、4、6、8、12周行X线、ECT、组织学等手段检测;远期在术后6、12个月行X线及组织学检查,评价骨缺损修复情况.结果 术后早期标本的大体观察和组织学观察看,在各个时间段,实验组的骨痂生成和血管化成度均优于对照组,从X线片和ECT可以看出,术后2周实验组和对照组比较差异无统计学意义,以后的各个时间段,实验组的骨修复效果均明显优于对照组;远期X线和组织学显示组织工程骨与兔桡骨牢固愈合,并开始塑形且出现髓腔再通,β-TCP在体内逐渐被吸收,自身架构消失.结论 早期采用筋膜瓣包裹的方法能够显著提高组织工程骨修复大段骨缺损的能力,远期组织工程骨可以完全修复兔大段骨缺损,形成正常骨组织并发挥功能. Abstract： Objective To investigate the long-term osteogenesis effects of large tissue engineered bone wraped up by blood supplied fascia. Methods Thirety -six New Zealand rabbits were subjected to operations to induce a 1.5 cm defect in rabbit' s two radiuses, which was filled subsequently with tissue engineered bone. The tissue engineering bone were wraped up by blood supplied fascia in rabbit's left radius, as the experimental group, the treatment wasn' t done to right radius, as the control group. At 2,4,6,8and 12 weeks after operation, X-ray examination, radionuclide bone scan and histological examination, were carried out to judge the osteogenesis effects. The repaired defects were evaluated by X-ray and histological examination at 6th , 12th month postoperation. Results In the early stage, gross observation and histology showed batter osteotylus generation and revascularization within 12 weeks postop-eratively experimental group than that in control group. Postoperative 2 weeks, X-ray and ECT displayed experiment group and control group do not have obvious difference, in each time later, the osteogenesis effects of the experiment group repairs was obviously superior to the control group. Longterm follow -up were performed by X -ray examination and histology at 6th, 12th month postoperatively revealed moulding of the new bones and medullary cavity recanalization, and the structure of β- TCP disappeared and gradually integrated into the new bones. Conclusions Early observation showed blood supplied fascia flap can accelerate the osteanagenesis process of tissue engineered bone. Long-term observation displayed tissue engineered bone is capable of total repair of large bone defect in rabbits by forming normal functional new bones.     

凝血酶肽复合人工骨修复兔桡骨大段骨缺损的实验研究

目的 探讨凝血酶肽(TP508)复合人工骨对兔桡骨大段骨缺损的修复效果。方法 随机选取新西兰大白兔42只,建立双侧桡骨1.5cm骨-骨膜缺损模型。随机 选取其中36只,将右侧作为实验侧,骨缺损处植入羟基磷灰石人工骨材料并注入TP508;左侧为对照组,仅植入上述人工骨材料;剩余6只作为空白对照组,骨缺损处不 植入任何物质。术后4周、8周、12周分批处死实验动物,对实验段桡骨进行X线及组织学分析。结果 术后4周、8周、12周实验组X线评分、组织学评分及骨缺损区 新生骨组织面积百分比均优于同期对照组(P均<0.05);术后12周空白对照组骨缺损区无骨连接形成,X线评分及组织学评分明显低于同期实验组及对照组(P均 <0.05)。结论 TP508在骨缺损修复过程中具有促进愈合作用,复合人工骨植骨对兔桡骨大段骨缺损的修复效果良好。     

半环槽外固定器建立山羊胫骨缺损模型

目的 探索半环槽外固定器建立山羊胫骨段缺损模型.方法 山羊9只分3组,制备胫骨中下段20%的骨膜和骨缺损,半环槽外固定器外固定,术后第5、10、15周分别处死1组动物.用放射学、组织学方法评价骨缺损修复情况.结果 术后山羊胫骨段缺损处固定稳定,5、10、15周骨缺损处未见骨修复,15周时见骨断端硬化,放射学和组织学检查未见骨形成.结论 应用半环槽外固定器建立组织工程用大段骨缺损动物模型效果较好,山羊胫骨20%的骨膜和骨缺损不能自行修复.     

珊瑚羟基磷灰石植入修复良性骨肿瘤缺损

目的: 观察无机材料珊瑚人工骨修复良性骨肿瘤刮除术后骨缺损的临床疗效.方法: 将天然珊瑚经特殊处理加工成羟基磷灰石,作为骨修复的填充材料,植入骨肿瘤刮除术后骨缺损区.共治疗25例患者,均为良性骨肿瘤.缺损范围最大10 cm×3.5 cm×2 cm,最小0.8 cm×0.5 cm×0.5 cm.结果: 所有病例术后查血生化正常,伤口愈合良好,无异物排出现象.X线显示:1月后管状骨周围有骨痂生长,3月植入的珊瑚骨密度开始逐渐降低,而珊瑚骨之间的空隙密度增高,4月达到临床愈合,18月珊瑚羟基磷灰石基本完全吸收.结论: 天然珊瑚制成羟基磷灰石具有良好的生物相容性和骨传导性,而且生物降解时间延长,基本与骨形成达到同步,是一种较为理想的骨缺损修复材料.     

消旋聚乳酸/羟基磷灰石/脱钙骨基质人工骨修复兔桡骨大段骨缺损的实验研究

目的研制理想的,能够修复大段骨缺损的人工骨材料。方法采用乳液共混法将消旋聚乳酸(PDLLA)、羟基磷灰石(HA)、脱钙骨基质(DBM)结合,制成PDLLA/HA/DBM人工骨,并将PDLLA/HA/DBM和PDLLA进行兔桡骨大段骨缺损修复的对比研究。术后2、4、8和12周时摄X片及病理形态学观察及新骨形成定量分析。结果PDLLA/HA/DBM人工骨内新骨形成量明显多于PDLLA及空白对照组(P<0.01),且能够有效修复骨缺损。结论PDLLA/HA/DBM人工骨能促进长骨大段骨缺损的修复,是一种较理想的骨修复材料。     

同种异体兔骨髓间充质干细胞复合纳米晶胶原基骨修复胫骨缺损的实验研究

目的 探讨同种异体兔骨髓间充质干细胞(marrow mensenchymal stem cells,MSCs)与纳米晶胶原基骨(nano-hydroxyapa-tite/collagen,NHAC)修复材料构建的组织工程骨修复兔胫骨缺损的可行性.方法 24只新西兰大白兔胫骨中段形成10mm长的骨缺损,右侧骨缺损处植入组织工程骨作为实验组,左侧骨缺损处植入单纯NHAC作为对照组.术后3、6、9、12wk分批处死动物,行一般情况、缺损区大体观察、X线、组织学染色分析等指标检测,行统计学分析,比较各组修复骨缺损的效果.结果 24只新西兰大白兔均进入结果分析.①术后一般情况:各组兔术后恢复及进食均正常,伤口无炎症反应,愈合良好.②大体观察:实验组术后6wk骨缺损部分修复,9、12wk骨缺损完全修复,3、6、9、12wk骨缺损修复情况明显好于对照组.③X线:实验组缺损区术后3wk可见有骨痂生长,9wk骨缺损基本修复,对照组术后12wk缺损区基本修复,各观察时间点实验组骨缺损修复情况明显好于对照组.④组织学染色:实验组缺损区新生类骨样组织、编织骨和板状骨出现的时间较对照组早,并且不经软骨介导即可直接成骨,而对照组以"爬行替代"方式修复骨缺损.结论 同种异体兔MSCs复合NHAC修复骨缺损的能力较单纯NHAC强且迅速,能够对大段骨缺损进行快速有效的修复.     

同种异体兔骨髓间充质干细胞复合纳米晶胶原基骨修复胫骨缺损的实验研究

目的探讨同种异体兔骨髓间充质干细胞（marrow mensenchymal stem cells，MSCs）与纳米晶胶原基骨（nano—hydroxyapatite／collagen，NHAC）修复材料构建的组织工程骨修复兔胫骨缺损的可行性。方法24只新西兰大白兔胫骨中段形成10mm长的骨缺损，右侧骨缺损处植入组织工程骨作为实验组，左侧骨缺损处植入单纯NHAC作为对照组。术后3、6、9、12wk分批处死动物，行一般情况、缺损区大体观察、X线、组织学染色分析等指标检测，行统计学分析，比较各组修复骨缺损的效果。结果24只新西兰大白兔均进入结果分析。①术后一般情况：各组兔术后恢复及进食均正常，伤口无炎症反应，愈合良好。②大体观察：实验组术后6wk骨缺损部分修复，9、12wk骨缺损完全修复，3、6、9、12wk骨缺损修复情况明显好于对照组。③X线：实验组缺损区术后3wk可见有骨痂生长，9wk骨缺损基本修复，对照组术后12wk缺损区基本修复，各观察时间点实验组骨缺损修复情况明显好于对照组。④组织学染色：实验组缺损区新生类骨样组织、编织骨和板状骨出现的时间较对照组早，并且不经软骨介导即可直接成骨，而对照组以“爬行替代”方式修复骨缺损。结论同种异体兔MSCs复合NHAC修复骨缺损的能力较单纯NHAC强且迅速，能够对大段骨缺损进行快速有效的修复。     

携载BMP4基因的核壳结构纳米粒子修复兔桡骨临界骨缺损的研究

目的 将改性的壳聚糖包裹骨形态发生蛋白4(BMP4)质粒形成核壳结构,以明胶海绵为此纳米粒子载体,探究其对兔桡骨临界骨缺损的修复作用.方法 新西兰大白兔24只,每只兔子的左右前肢按随机法分为负载BMP4质粒核壳结构组(TACS@EG-HBC/pBMP4/G组)、负载BMP4质粒核结构组(TACS/pBMP4/G组)和对照组.在双侧桡骨制备长约18 mm的完全性临界骨缺损,分别植入含有TACS@EG-HBC/pBMP4或TACS/pBMP4的明胶海绵,对照组仅植入单纯的明胶海绵.术后2、4、8、12周取标本做分别处理,检测缺损部位的大体标本、骨密度及骨矿物质含量、X线、HE染色、免疫组化法、生物力学.结果 24只兔子均纳入分析,术后切口未见感染、化脓等症状.大体标本示:TACS@EG-HBC/pBMP4/G组骨缺损完全骨化修复;骨密度及骨矿物质含量显著高于TACS/pBMP4/G组和对照组(P＜0.05);X线示:术后12周骨缺损处已完全修复,骨髓腔已再通;HE染色结果显示:新生骨小梁相互连接成板层骨;免疫组化结果显示:BMP4棕色蛋白染色明显;生物力学测定结果显示:所形成的新生骨与正常骨组织生物力学差异无统计学意义.结论 TACS@EG-HBC/pBMP4/G具有良好的安全性和成骨能力.     

Repair of sheep metatarsus defects by using tissue-engineering technique

Tissue-engineering bone with porous β-tricalcium phosphate (β-TCP) ceramic and autologous bone marrow mesenchymal stem cells (MSC) was constructed and the effect of this composite on healing of segmental bone defects was investigated. 10-15 ml bone marrow aspirates were harvested from the iliac crest of sheep, and enriched for MSC by density gradient centrifugation over a Percoll cushion (1.073g/ml). After cultured and proliferated, tissue-engineering bones were constructed with these cells seeded onto porous β-TCP, and then the constructs were implanted in 8 sheep left metatarsus defect (25 mm in length) as experimental group. Porous β-TCP only were implanted to bridge same size and position defects in 8 sheep as control group, and 25 mm segmental bone defects of left metatarsus were left empty in 4 sheep as blank group. Sheep were sacrificed on the 6th, 12th, and 24th week postoperatively and the implants samples were examined by radiograph, histology, and biomechanical test. The 4 sheep in blank group were sacrificed on the 24th week postoperatively. The results showed that new bone tissues were observed either radiographic or histologically at the defects of experimental group as early as 6th week postoperatively, but not in control group, and osteoid tissue, woven bone and lamellar bone occurred earlier than in control group in which the bone defects were repaired in “creep substitution” way, because of the new bone formed in direct manner without progression through a cartilaginous intermediate. At the 24th week, radiographs and biomechanical test revealed an almost complete repair of the defect of experimental group, only partly in control group. The bone defects in blank group were non-healing at the 24th week. It was concluded that engineering bones constructed with porous β-TCP and autologous MSC were capable of repairing segmental bone defects in sheep metatarsus beyond “creep substitution” way and making it healed earlier. Porous β-TCP being constituted with autologous MSC may be a good option in healing critical segmental bone defects in clinical practice and provide insight for future clinical repair of segmental defect.     

Repair of Rabbit Femoral Defects with a Novel BMP2-derived Oligopeptide P24

In this study, the bioactivity of a novel BMP2-derived oligopeptide P24 was investigated by using the model of rabbit femoral defect after loaded in the biodegradable poly （lactic acid / glycolic acid / asparagic acid-co-polyethylene glycol） （PLGA-[ASP-PEG]）. A 1.5-cm unilateral segmental bone defect was created in the left femoral diaphysis in each of the 30 new zealand white rabbits. The defects of 18 legs filled with BMP2-derived peptide P24 combined with PLGA-[ASP-PEG] scaffold serves as the experimental group, and the defects in the rest 12 rabbits filled with （PLGA-[ASP-PEG]） without P24 as control group. The bone-repairing capability in the target region of the two group was grossly, radiologically, histopathologically and biomechanically evaluated 4, 8 and 12 weeks after the operation. Our results showed that in each group, primary healing of incision was achieved in the two groups. Radiographically, in experimental group, defects were filled with induced callus within 8 weeks, and a cortical bone-like structure was observed in some animals at the 12th week. According to the standardized stage of bone defect repair, 9 （64.28%） achieved grade-4 healing. In contrast, little bone formation was seen in the defects even 12 weeks after the operation, and 5 （62.50%） had grade 0 healing in this group. Histologically, tissue engineering material was mostly absorbed and cartilage was found around implants in the experimental group at the 4th week; 8 weeks after operation, the engineering material was completely absorbed, and formation of woven bone was observed and typical trabecular bone structure could be seen. In control group, 8 weeks after operation, the defect was filled with fibrous tissues, and no bone-like structure was observed. Statistical analysis showed very significant difference in biomechanical indicators between the two groups （P〈0.05）. It is concluded that new oligopeptide P24 can induce excellent bone regeneration and promote bone repair.     

BMP-9基因修饰的骨髓基质细胞促进山羊骨缺损愈合

目的观察BMP-9基因修饰的骨髓基质细胞(marrow stromal cells,MSCs)治疗山羊骨缺损的效果。方法 12只12～18月龄中国青山羊,股骨中段建立临界骨缺损模型,按照配对比较法随机分为2组,缺损区域明胶海绵填充,实验组加入预先准备好的BMP-9腺病毒感染的MSCs悬液,对照组加入GFP腺病毒感染的MSCs悬液,术后1 d、2、4、8、12、20周行X线片检查,20周时进行CT检查,处死动物,行大体标本、组织学染色和生物力学的检测。结果 X线片显示实验组较对照组骨痂形成更早更多,大体标本、组织学染色显示实验组的新生骨更加成熟,CT显示实验组新生骨塑形更好,生物力学检测显示实验组新生骨痂的力学强度更强。结论 BMP-9基因修饰的MSCs可以有效地促进山羊股骨骨缺损的愈合。